1. Field of the Invention
The present invention relates to a recombinant microorganism producing quinolinic acid, and a method for producing quinolinic acid using the same.
2. Description of the Related Art
Quinolinic acid, which is also called 2,3-pyridine-dicarboxylic acid, has a wide variety of applications as a precursor of synthetic chemicals in the production of medicinal or agricultural chemicals, dyes or the like.
Quinolinic acid can be prepared by chemical or biological synthetic methods. because of using non-renewable materials derived from petroleum as the raw material, chemical synthetic methods are greatly influenced by environmental problems, oil prices or the unit cost of petroleum extraction.
For Example of a representative biological synthetic method, a method of producing quinolinic acid in an E. coli strain is disclosed that expression of genes encoding L-aspartate oxidase (NadB) and quinolinate synthase (NadA) is enhanced by cloning a plasmid having different copy numbers of the two genes into E. coli, of which quinolinate phosphoribosyltransferase activity is eliminated (Eur. J. Biochem. 175, 221-228 (1988), DE3826041). In this regard, the concentration of quinolinic acid produced is as low as 500 mg/L or less. The first reason of this low production of quinolinic acid is transcriptional suppression by NadR, which is a NAD-related transcriptional repressor of nadB encoding L-aspartate oxidase and nadA encoding quinolinate synthase. The second reason is feedback inhibition of L-aspartate oxidase, NadB protein by NAD. And the third reason is a weak biosynthetic pathway from carbon sources to L-aspartic acid in E. coli. 
To solve the first reason, in Korean Patent laid-open No. 10-2012-0082673, a microorganism strain was used to increase the production of quinolinic acid to 5.5 g/L, wherein the promoter regions of L-aspartate oxidase (NadB) and quinolinate synthase (NadA), which are the quinolinate synthesis genes suppressed at the transcriptional level, were substituted with a constitutive promoter, and L-aspartic acid biosynthetic pathway is enhanced.
The biosynthetic pathway of quinolinic acid from L-aspartic acid by a biological synthetic method is as shown in the following reaction scheme:
1) L-aspartate Oxidase (NadB)L-aspartic acid+oxygen<=>hydrogen peroxide+α-iminosuccinate+H+
2) Quinolinate Synthase (NadA)α-iminosuccinate+dihydroxyacetone phosphate<=>quinolinic acid+phosphate+2H2O
α-iminosuccinate as an intermediate of the quinolinic acid biosynthetic pathway is an unstable substance, and is converted to oxaloacetate by natural deamination reaction in cells (THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 257, No. 2, Issue of January 25. pp. 626-632, 1982). In general, when an unstable intermediate metabolite is used as a substrate, the low collision frequency between the substrate and enzyme produces a large amount of by-products. However, until now, there has been no attempt to solve the above problem regarding the production of quinolinic acid.
On the other hand, a fusion protein technology of linking heterogeneous enzymes or proteins via an amino acid linker to express as a single protein, has heen used for various purposes, such as increasing the protein expression level by linking a protein showing a low level-expression with a protein showing a high level-expression, increasing the protein purification yield by preparing a fusion protein linked with a tag, or the like.
Design of the linker is known to be important in the preparation of fusion proteins. In general, a helical linker having an alpha-helix structure or a flexible linker having structural flexibility has been frequently used, and for example, various linkers can be designed and used by the combination of the two linkers according to the characteristics of the fusion protein to be achieved.
The present inventors have tried to solve the reduced reaction due to unstable metabolites by expression of a fusion protein of NadB and NadA linked via various types of amino acid linkers, and then they found that quinolinic acid can be produced in a high yield by expression of the fusion protein, compared to the yield of the conventional biological production method, thereby completing the present invention.